For decades, many proteins useful to humans or animals have been isolated from plants. With the advent of genetic engineering technology, a plant could be modified to produce human, animal, viral, bacterial, or fungal proteins. Transgenic plants offer the potential to be one of the most economical systems for large-scale production of proteins for industrial, pharmaceutical, veterinary and agricultural use. Advantages of plant systems include the low cost of growing a large biomass, easy scale-up (increase of planted acreage), natural storage organs (tubers, seeds), and established practices for efficient harvesting, transporting, storing, and processing of the plant. Recombinant proteins can be targeted to seeds allowing stable storage of the recombinant proteins for extended periods. Plants offer advantages over other production systems since some proteins may be used without extensive purification, because for many applications, plant material is used directly as a food source or feed stock.
Examples abound for expression of foreign genes in plants Benfey P N, Chua N—H: Regulated genes in transgenic plants. Science 244:174–181 (1989); Fisk H J, Dandekar A M: The introduction and expression of transgenes in plants. Scientia Hort. 55:5–36 (1993). In general, the expression of these foreign genes has been aimed at benefiting the consumer through plant improvement by: a) expressing antifungal compounds or growth factors; b)improving agronomic traits such as fruit ripening or nutritional contents or c) inducing sterility in the context of creating hybrid plants. It is also feasible to express in plants heterologous genes that encode high value products, a technology currently being explored by several plant biotechnology companies and university laboratories. In many cases, expression in plants could be the system of choice because of such inherent advantages as cost relative to that of animal tissue culture, and the concern that prokaryotic or yeast expression systems may not be capable of correct glycosylation and other post-translational processing steps required for proper function of the expressed protein Pen J, Sijmons P C, van Ooijen A J J, Hoekema A: Protein production in transgenic crops: Analysis of plant molecular farming. Industrial Crops Production. Elsevier, Amsterdam. pp. 241–250 (1993B). Thus, there is a need to improve such systems for increased efficiency of expression of the protein and to lower production costs. Among representative efforts to achieve such goals is the Goodman et al patent assigned to Calgene, U.S. Pat. No. 5,550,038, which discloses constructs for expression of physiologically active mammalian proteins in plant cells. The isolation and purification procedure for the mammalian peptides disclosed there is to preparation from frozen tobacco tissue obtained from tissue culture which is ground in liquid nitrogen, centrifuged, washed with ethylene glycol and dialyzed overnight in dialysis buffer to obtain γ-IFN.
Vanderkerckhove et al, assigned to Plant Genetic Systems N.V, at U.S. Pat. No. 5,487,991, discloses a method for producing polypeptides by cultivating a plant whose genome contains recombinant DNA. Recovery of transgenic proteins is accomplished by harvesting seeds from cultivated plants, cleaving out the peptide of interest and recovering the peptide of interest in a purified form. The recovery of the active polypeptides involves homogenizing the entire seed in dry ice and extraction with hexane, extraction with high salt buffer and dialysis against distilled water and precipitating the contaminating globulins. Further purification is accomplished by gel-filtration chromatography, and finally ion-exchange chromatography.
The extraction process shown at PCT WO92/010402 by Willmitzer et al and assigned to Novo Nordisk provides for homogenizing plant tissue and use of extraction buffer, filtration and centrifugation.
In PCT WO95/14099 by Rodriguez et al, assigned to the University of California, methods for production and secretion of heterologous proteins in plants are discussed wherein malting monocot plant seeds is disclosed to stimulate heterologous protein production in cereal seeds, causing conversion of the endosperm to maltose and germination of the seeds. The chimeric gene includes a transcriptional regulatory region inducible during seed germination, a DNA sequence encoding a protein of interest and further contains a signal sequence linked to the transcriptional regulatory region effective to facilitate secretion of the protein across the aleurone or scutellar epithelium layer into the endosperm. In one embodiment, the embryos and endosperm may be separately steeped in 55° F. water for 48 hours followed by four day germination in bins or drums with inducement of a promoter or addition of plant hormones. This is because expression in the embryo was poor unless different conditions were used to cause induction of the protein in the embryo versus the endosperm. The embryo and endosperm portions are then mixed and mashed.
Factors that can be manipulated to control levels of expression are the presence of transcriptional modification factors such as introns, polyadenylation signals and transcription termination sites. Intron sequences within the gene of interest also may increase its expression level by stabilizing the transcript and allowing its effective translation. Many plant genes contain intron sequences exhibiting this positive impact on expression (Callis J, Fromm M, Walbot V: Introns increase gene expression in cultured maize cells. Genes and Development 1:1183–1200 (1987)) including for example some of the plant ubiquitin genes Christensen A M, Sharrock R A, Quail P H: Maize polyubiquitin genes: structure, thermal perturbation of expression and transcript splicing, and promoter activity following transfer to protoplasts by electroporation. Plant Mol. Biol. 18:675–689 (1992); Christensen A M, Sharrock R A, Quail P H: Maize polyubiquitin genes: structure, thermal perturbation of expression and transcript splicing, and promoter activity following transfer to protoplasts by electroporation. Plant Mol. Biol. 18:675–689 (1992) and the Adh2 gene (Callis, supra). At the translational level, factors to consider that affect expression level of foreign genes are the ribosomal binding site and the codon bias of the gene [Cornejo M, Luth D, Blankenship K, Anderson O, Blechl A: Activity of a maize ubiquitin promoter in transgenic rice. Plant Mol. Biol. 23: 567–581 (1993) and references therein]. High-level expression of a gene product which accumulates in the cytoplasm may result in toxicity to the plant cell. Therefore, sequestering the protein into a compartment (organelle) or transporting it to the extracellular matrix may allow higher expression levels. Efforts are being made to understand plant protein targeting (An G, Mitra A, Choi H K, Costa M A, An K, Thornburg R W, Ryan C A: Functional analysis of the 3′ control region of the potato wound-inducible proteinase inhibitor II gene. Plant Cell 1:115–122 (1989); Jones R L, Robinson D G: Protein secretion in plants. New-Phytol. New York, N.Y.: Cambridge University Press. April 1989. v. 111 (4) p. 567–597. ill) and proteins can be effectively targeted to the mitochondrion, the chloroplast, the vacuole, peroxisomes or the cell wall. The specific choice of where to target will depend on the nature of the protein of interest and the specific need. Insertion of a construct at different loci within the genome has been observed to cause variation in the level of gene expression in plants. The effect is believed to be due at least in part to the position of the gene on the chromosome, producing individual isolates with different expression levels (Staehelin L A, Moore I: The plant golgi apparatus: Structure, functional organization and trafficking mechanisms. Ann. Rev. Plant Physiol. Plant Mol. Biol. 46:261–288 (1995)).
One of the critical factors in expression of protein in plants is the choice of transcriptional promoters used. The range of available plant compatible promoters includes tissue-specific and inducible promoters. Some of the better documented constitutive promoters include the CaMV 35s promoter and its tandem arrangement, as described in European patent application number 0 342 926, and the ubiquitin promoter, as disclosed in Quail et al, assigned to Mycogen Plant Science, Inc. U.S. Pat. No. 5,510,474.
The invention here improves on what has been known through the determination that the germ can be separated from the endosperm, not for enriching the endosperm fractions, but to be used separately for recovery of protein and high activity obtained. The germ (which is the term for the embryo used in commercial plant production) is surprisingly found to have an enhanced concentration level of protein on a dry weight basis. Thus, only a small amount of tissue is needed to provide high levels of protein expression. This provides for considerable cost savings, as the separated endosperm and other parts of the seed can be sold for food, feed, and other commercial processing and the much smaller germ material as opposed to the entire seed is used, producing more protein per material processed. Commercial production of protein from plant biofactories has not previously considered use of the germ as the protein source; instead those in the field desired to use the entire seed as the source of protein, since presumably it would yield higher amounts of protein available for the commercial process to which it would be applied. Instead, the inventors have found that the germ not only can be the source of the protein, but is a more desirable source of the protein.
The protein can be extracted from the germ, or the germ tissue can be used directly in the commercial process. In the latter situation, cost of the protein production is even further decreased. In one embodiment, laccase can be produced in plants and the germ used as the source of the enzyme. The germ can be used directly in commercial processes where laccase is useful.
Further, expression of protein can be directed to the germ, further enhancing protein recovery. For example, the ubiquitin promoter, believed to have been constitutively expressed, greatly increases expression of protein in the germ.
The present invention relates, in general, to a novel method of catalyzing in vitro reactions using transgenic plant germ tissues that over-express a desired protein. More specifically, the present invention relates to the enzymatic polymerization of lignin based compositions with laccase enzymes. The present invention also pertains to a method of enhancing the biochemical availability of laccase.
Laccases (E.C. 1.10.3.2) are polyphenol oxidizing enzymes. They are blue copper oxidases in that they contain a copper atom or atoms as a prosthetic group. Together, laccases and the aromatic residues of lignin can produce reactive groups in the lignin molecule in a kind of radical phenol-oxidation reaction, whereby these groups then polymerize and/or cross-link in secondary reactions. Laccases are known from plants, fungi and animal sources. The main source of laccase for commercial purposes is fungi such as the basidiomycetes Phanerochaete chrysosporium and Trametes versicolor. Other fungi producing laccase include Aspergillus, Neurospora, Botrytis, Polyporus, Pleurotus, Philota, Podospora, Collybia and Rhizoctonia. 
The uses of laccases are numerous. Examples include catalyzing the oxidation of compounds such as o,p-diphenols, aminophenols, polyphenols, polyamines, and inorganic ions (Yaropolov et al. 1994. Applied Biochemistry and Biotechonology 49:257–280). The use of laccases as marker enzymes in enzyme immunoassays (EIA) has also been discussed, as well as their use in the oxidation of steroids and synthesis of vinblastine, a cytostatic compound used in treating malignant diseases. Laccase can also be used in gelling reactions. Bolle and Aehle in a U.S. Pat. No. 6,217,942 to Genencor International, Inc. describe a method of using laccase for the production of a coated article from the waste liquors produced by the pulp and paper industry. The method comprises: (a) preparing a solution of lignin, said solution preferably comprising lignin sulfonate, (b) mixing the lignin solution with a phenol oxidizing enzyme, preferably laccase, (c) incubating the mixture under conditions sufficient to form a solution of desired viscosity, (d) contacting or spreading the mixture from step (c) on an article to be coated, and (e) allowing the film to set onto the article by subjecting the article to conditions and for a time sufficient to form a film on the surface of the article.
A common use of laccase is in connection with the paper and pulp industry. Lignin is a rigid organic polymer and harsh physiochemical conditions must be used to attack or modify the substance. Naturally occurring white rot fungi destroy lignin using laccases and peroxidases. In plants, laccases are localized in woody tissues and cell walls of herbaceous species and it is believed it participates in lignin biosynthesis. It is involved in breaking down lignin as well as creating lignin polymers. It is also especially useful as a “biological glue” or adhesive or binder when manufacturing glued wood products. Such products include construction and industrial plywood, oriented strand board, particleboard and medium density fiberboard.
Currently, the adhesive mostly used is either a urea-formaldehyde type or a phenol-formaldehyde resin. There are disadvantages associated with use of formaldehyde in producing such products. Processing and end use monitoring are required as the levels of formaldehyde cannot exceed certain controls. Thus, there has been considerable interest in using such natural alternatives as laccase. It is reported that more than 1.2 million metric tons of adhesive resin solids are used to bond glued wood products in the United States. Which adhesive is used is driven by cost per unit of production, process compatibility and end-use durability (Technical and Market Opportunities for Glued Wood Products” Adhesive Age May 31, 1996 V39, N6 p.609).
An example of such a process is described by Kharazipour et al in U.S. Pat. No. 5,505,772 and by Olesen et al. at U.S. Pat. No. 5,618,482. In general, fibers and chips from wood or wood-like materials are defibrated by mechanical, steam, or other process. Laccase is then brought into contact with the material in a solution that may contain various auxiliary elements. Since laccase is a large molecule, a mediator may be utilized to aid the enzyme activity in penetrating the wood and may be added to the solution. The mix is incubated and may then be shaped into formed boards.
There has also been some interest in the field for enhancing laccase activity and/or stability in in-vitro reactions. One example is described by Schneider et al in a U.S. Pat. No. 5,795,855 to Novo Nordisk A/S. This patent discusses methods for enhancing laccase activity using organic chemical compounds. These chemical compounds consist of at least two aromatic rings, of which at least one ring is substituted with one or more of the following atoms: nitrogen, oxygen, and sulfur; and which aromatic rings may furthermore be fused rings. They report that the addition of such enhancers leads to a much faster bleaching of a dye (Direct Blue 1). It is obvious, therefore, that enhancing the biochemical activity of laccase is a sought-after invention that could be useful for various industries. Laccase production in plants does that without the use of environmentally polluting chemicals.
The compound ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate), supplied by Boehringer Mannheim, has been proposed to act as a redox mediator for oxidation of non-phenolic lignin model compounds (Bourbonnais and Paice. 1990. FEBS Lett. 267: 99–102). Studies on demethylation and delignification of kraft pulp by a laccase showed that the extent of partial demethylation by laccase was increased in the presence of ABTS (Bourbonnais and Paice. 1992. Appl. Microbiol. Biotechnol. 36: 823–827). Other accelerators or enhancers have also been described. Metal ions, phenolic compounds such as 7-hydroxycoumarin, vanillin and p-hydroxybenzenesulfonate, have been described as compounds capable of enhancing bleaching reactions (WO 9218683, WO 9218687).
An effect of plant substances on enzyme performance has been reported by Lacki and Duvnjak (1999) Stability of polyphenol oxidase from the white-rot fungus Trametes versicolor in the presence of canola meal. Acta Biotechnol. 19:2, 91–100.). The authors found that canola meal improves the thermal stability of a polyphenol oxidase. The authors, however, did not report any enhancement in the activity or availability of the polyphenol oxidase in the presence of canola meal.
U.S. Pat. No. 5,543,576 presents a method of catalyzing in vitro reactions using transgenic seeds containing enhanced amounts of enzymes. The method involves directly adding transgenic seeds, preferably in a ground form, to a reaction mixture. The process uses whole seeds and does not disclosure use of germ plant tissue as source of the enzyme. By use of the germ, less tissue is needed to obtain higher concentration on a dry weight basis, providing cost advantages, recovery of costs from sale of the other portions of the seed, and less tissue which needs to be stored, handled and processed.
Thus it is an object of the invention to decrease cost in production of commercial protein through using germ of seed expressing the protein in commercial processes.
An object of the invention is to use seed for production of commercial protein more efficiently.
It is another object of the invention to use the germ portion of the seed for production of commercial protein while retaining high activity of the recombinant protein.
Another object of the invention is to increase concentration of heterologous protein in plant tissue on a dry weight basis by using the germ as the source of the protein.
A still further object of the invention is to direct expression of heterologous protein in a seed to the germ or embryo portion of the seed.
An additional object of the invention is to provide a safe method for storing and handling heterologous proteins produced in plants. The germ matrix protects the enzyme from degradation due to endogenous protease inhibitors and the desiccated form of the germ. The germ matrix stabilizes the enzyme while still leaving it available to react with the substrates.
Yet another object of the invention is to increase concentration of heterologous laccase produced in plant tissue on a dry weight basis by using the germ as the source of laccase.
Another object of the invention is to use germ as the source of heterologous laccase by addition of the germ in a commercial process.
A further object of the invention is to overcome limitations on the amount of laccase available in commercial processes when the laccase is produced in plants due to the discovery that a certain amount of laccase cannot be removed from plant cells. Instead, the germ tissue is used in the process, making all the laccase contained in the tissue available.
The foregoing objectives and others will become apparent in the description below.